1. Field of the Invention
The present invention relates to a fabrication method of a microfluidic circuit employed in biochemical tests such as of DNA, protein, cells, blood, or the like. The present invention also relates to a microfluidic circuit effective for use in μTAS (Micro Total Analysis System) employed in chemical synthesis, environment analysis, or the like.
2. Description of the Related Art
A microfluidic circuit allows a series of experimental operations carried out in a laboratory to be implemented in a chip that is approximately 2 cm square and 2 mm in thickness. The microfluidic circuit provides various advantages such as: only a small amount of samples and reagents is required; costs are low; reaction speed is fast; high-throughput tests can be performed; and results can be obtained immediately at the site where the sample was taken.
A plan view of an exemplary microfluidic circuit is shown in FIG. 1. This circuit is directed to a hepatic function test. Referring to FIG. 1, approximately 10 μL of blood is introduced into the circuit through a sample injection inlet 1. The blood is separated into blood cells and blood plasma by centrifugation. Only the blood plasma is delivered to a blood plasma retaining chamber 2. In a weighing chamber 3, the amount of blood plasma is measured, and the blood plasma is transferred to a blending chamber 7. Then, the reagent stored in a reagent retaining chamber 4 is blended. After the blood plasma and the reagent are mixed in a mixing chamber 5, examination of the hepatic function is conducted at a measuring chamber 6. The measurement is carried out by directing a laser beam of short wavelength and detecting the light absorption with a photodiode. In this manner, a series of operations from the preprocessing of the sampled blood to the aforementioned measurement can be carried out within the microfluidic circuit to effect tests on various items such as γ-GTP, AST (GOT), ALT (GPT), lactate dehydrogenase (LDH), and the like (refer to “All electronic appliance and machinery companies compete for the biochip market”, Nikkei Biobusiness, December 2003, pp. 42-43).
A fabrication method of a conventional microfluidic circuit is shown in FIGS. 4A-4K. In accordance with this method, the microchannel (flow channel), the reagent retaining chamber and the like of the microfluidic circuit are formed by microfabrication techniques based on the combination of photolithography, etching, and molding. First, a silicon substrate 41 is heated under an oxygen ambient to form an SiO2 film 42 on silicon substrate 41 (FIG. 4A). Then, a resist 43 is formed on SiO2 film 42 (FIG. 4B). For the resist, resist containing polymethacrylic acid ester such as polymethyl methacrylate (PMMA) as the main component, or a chemical amplification resist having sensitivity to ultraviolet (UV), is employed.
Then, a mask 44 is arranged on resist 43, and UV 45 is directed through mask 44 (FIG. 4C). Mask 44 is constituted of an UV absorption layer 44b formed in accordance with the arrangement and shape of the microchannel, the reagent retaining chamber and the like in the microfluidic circuit to be fabricated, and a translucent base 44a. Quartz glass or the like is employed for translucent base 44a. Chromium or the like is employed for absorption layer 44b. In the case where a positive resist is employed, irradiation of UV 45 causes resist 43b alone to be exposed and changed in property by the function of absorption layer 44b. Resist 43b is removed by development to leave resist 43a (FIG. 4D). In contrast, when a negative resist is employed, the exposed portion is left and the non-exposed portion is removed. Therefore, a mask pattern of a version opposite to that of a positive resist is employed.
Next, plasma etching or wet etching is effected using resist 43a as a mask (FIG. 4E), and then resist 43a is removed (FIG. 4F). A metal film is formed by vapor deposition or the like on the obtained SiO2 film 42a (FIG. 4G). Silicon substrate 41 and SiO2 film 42a are removed by wet etching or mechanical peeling to result in a mold 46 (FIG. 4H). Then, injection molding or the like with molten plastic is conducted using mold 46 (FIG. 4I) to fabricate a light transmissive substrate 47a (FIG. 4J). Polyethylene terephthalate, for example, is used for the plastic. Finally, light transmissive substrate 47a is bonded with a corresponding light absorptive substrate 47b. Thus, a microfluidic circuit 47 including a microchannel can be obtained (FIG. 4K) (refer to the aforementioned “All electronic appliance and machinery companies compete for the biochip market”, Nikkei Biobusiness, December 2003, pp. 42-43).
The method of bonding light transmissive substrate 47a with corresponding light absorptive substrate 47b includes heat fusion through thermal pressing or ultrasonic waves, a method employing an adhesive, or the like. The heat fusion method is disadvantageous in that the microchannel will be readily deformed by excessive heat, and that the bioactive substance fixed may be adversely affected to cause functional inhibition. The method using an adhesive is disadvantageous in that the excessive adhesive material may ooze into the microchannel to block the microchannel or contaminate the inner wall.
There is known a laser bonding method as an improvement of the disadvantages set forth above, as disclosed in Japanese Patent Laying-Open Nos. 2005-074796 and 2000-218698. A conventional laser bonding method is shown in FIG. 2. A laser beam 22 output from a laser light source 21 is arranged to enter a contact face 26 of light transmissive substrate 24 and light absorptive substrate 25 perpendicularly. Light transmissive substrate 24 is formed of a material that allows transmittance of laser beam 22. Light absorptive substrate 25 is formed of a material that absorbs laser beam 22. By directing a laser beam 22 with laser light source 21 moved in the direction of the arrow, contact face 26 of light transmissive substrate 24 and light absorptive substrate 25 is fused by the exposed light and then cooled for bonding. Since a microchannel (not shown) is formed at the bottom of light transmissive substrate 24, irradiation of the microchannel with laser beam 22 during the fusion step, as for contact face 26, will cause the microchannel to be heated and deformed. Therefore, mask 23 is employed to prevent the microchannel from being irradiated with the laser beam.
This method of blocking light using a mask is disadvantageous in that a relevant mask must be provided, and an extra step of registration between the mask and the microchannel is required. The fabrication efficiency of microfluidic circuits is reduced to become a factor of increasing the cost. There is also a problem that the positioning between the mask and microchannel is readily deviated. Such deviation in positioning may cause heat deformation at the microchannel, leading to nonuniform bonding. This may become the cause of liquid leakage. It is therefore difficult to obtain an accurate measurement.